Modifications of peptide compositions to increase stability and delivery efficiency

ABSTRACT

The disclosed invention relates to methods of modifying peptide compositions to increase stability and delivery efficiency. Specifically, the disclosed invention relates to methods to increase the stability and delivery efficiency of protein kinase C (PKC) modulatory peptide compositions. A “therapeutic peptide composition” comprises a “carrier peptide” and a “cargo peptide.” A “carrier peptide” is a peptide or amino acid sequence within a peptide that facilitates the cellular uptake of the therapeutic peptide composition. The “cargo peptide” is a PKC modulatory peptide. Peptide modifications to either the carrier peptide, the cargo peptide, or both, which are described herein increase the stability and delivery efficiency of therapeutic peptide compositions by reducing disulfide bond exchange, physical stability, reducing proteolytic degradation, and increasing efficiency of cellular uptake.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/305,685, filed Nov. 28, 2011, which is a divisional of U.S.application Ser. No. 12/017,985, filed Jan. 22, 2008, now U.S. Pat. No.8,067,532, which claims the benefit of priority of U.S. ProvisionalPatent Application Nos. 60/881,419, filed Jan. 19, 2007 and 60/945,285,filed Jun. 20, 2007, all of which are incorporated herein by referencein their entirety.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Jan. 8, 2016, and named“091508-0257_SequenceListing.txt” (43000 bytes), the contents of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This application relates to compositions and methods to improve carrierof biologically active agents into cells in living tissue. Thecompositions and methods comprise a PKC modulatory peptide conjugated toa modified tat peptide that imparts improved plasma stability to theconjugate, allowing more efficient uptake of the PKC modulatory peptideinto cells.

BACKGROUND ART

Research has produced many peptides that have potential as therapeuticcompositions. Yet realizing and exploiting the full therapeuticpotential of peptides directed against intracellular targets has yet tobe achieved, for a variety of reasons. One of the most important ofthese is that most therapeutic peptides do not possess the ability tocross cell membranes to reach their therapeutic targets. One solution tothis problem is the use of carrier peptides that act to ferry a cargopeptide into a target cell.

There are a number of notable examples of carrier peptides which areeffective to facilitate the crossing of a target cell's membrane by acargo peptide. One example is a peptide sequence derived from the TATprotein of the HIV virus. See U.S. Pat. No. 6,316,003, which is herebyincorporated by reference in its entirety. Another well known carrierpeptide sequence is the “poly-Arg” sequence. See, e.g., U.S. Pat. No.6,306,993.

In many cases, the use of a disulfide bond to link the carrier and cargopeptides, producing the therapeutic peptide construct, is an effectivestrategy to solve the problem of targeting soluble peptides tointracellular targets. One theory explaining the usefulness of disulfidebonds holds that once the carrier-cargo construct enters a target cell,the two peptides can separate through disulfide bond reduction. Thisseparation in the intracellular environment may allow a greaterdiffusion of cargo peptides within the cell as opposed to other linkagemechanisms which maintain the carrier-cargo link. With this said,however, the administration of therapeutic peptides still suffers fromnumerous challenges, such as disulfide bond exchange, proteolyticdegradation and efficiency of cellular uptake. Methods directed tocontrolling these issues will increase the stability and potency oftherapeutic peptides.

One way to increase the potency of a therapeutic peptide comprising acarrier peptide disulfide bonded to a cargo peptide is to reducedisulfide bond exchange. Disulfide bond exchange reduces the amount of acarrier-cargo peptide construct in a given sample by allowing a carrierpeptide to exchange its cargo peptide for another carrier peptide, thusresulting in a carrier-carrier construct and a cargo-cargo construct.The carrier-only construct will have no therapeutic effect. Thecargo-cargo construct will have a tremendously reduced, if notcompletely eliminated effect, since the carrier peptide enables thedelivery of the cargo to its intracellular target. As such, the problemof controlling disulfide bond exchange is important to maximizing thetherapeutic potential of a carrier-cargo peptide construct.

Another problem facing the use of therapeutic peptides is proteolyticdegradation. Peptides are notoriously unstable molecules and frequentlylabile to proteolytic attack when administered to a subject. Labilecarrier peptides which degrade upon administration will reduce or eveneliminate the efficacy of the cargo peptide because the cargo dependsupon the carrier peptide to reach the intracellular target. Thus,methods to control or eliminate the labile nature of therapeuticpeptides are also important to maximizing a carrier-cargo peptide'stherapeutic potential.

Increasing the efficiency of cellular uptake of a therapeutic peptide isyet another problem which can reduce the efficacy or potency of atherapeutic peptide. Optimization of carrier peptide sequences andplacement relative to the cargo peptide provide methods for increasingthe stability and potency of therapeutic peptide constructs.

DISCLOSURE OF THE INVENTION

The disclosed invention relates to methods of preparing a therapeuticpeptide composition comprising a carrier peptide and a PKC activitymodulating cargo peptide, whereby the resulting therapeutic peptidecompositions have increased stability and potency relative to anunmodified therapeutic peptide. One embodiment of the invention is amethod of decreasing disulfide bond exchange in a therapeutic peptidecomposition, comprising providing a therapeutic peptide composition,which comprises a carrier peptide comprising a first cysteine residueand a PKC activity modulating cargo peptide comprising a second cysteineresidue, wherein the carrier peptide and the cargo peptide are linked bya cysteine-cysteine disulfide bond between the first and second cysteineresidues, and introducing at least one aliphatic residue immediatelyproximate to the first or second cysteine residues, or both, whereby therate of disulfide bond exchange is decreased relative to an unmodifiedtherapeutic peptide composition.

Another embodiment of the disclosed invention related to a method ofdecreasing proteolytic degradation of a therapeutic peptide composition,comprising providing a therapeutic peptide composition, which comprisesa carrier peptide and a PKC activity modulating cargo peptide, andwherein the carrier peptide is linked to the cargo peptide, identifyinga proteolytically labile site on the carrier peptide, the cargo peptide,or both peptides, and modifying the amino acid sequence at the labilesite such that the rate of proteolytic degradation at the site isdecreased relative to an unmodified therapeutic peptide composition.

Another embodiment of the disclosed invention relates to a method ofincreasing plasma stability of a therapeutic peptide composition,comprising providing a therapeutic peptide composition, which comprisesa carrier peptide and a PKC activity modulating cargo peptide, andwherein the carrier peptide is linked to the cargo peptide, modifyingthe amino terminal, carboxy terminal or both residues of the carrierpeptide, the cargo peptide, or both, such that the plasma stability ofthe therapeutic peptide composition is increased relative to anunmodified therapeutic peptide composition.

Compositions are also contemplated disclosed herein. One embodiment ofthe disclosed compositions comprises a protein kinase C (PKC) modulatorypeptide composition, comprising a PKC modulatory peptide covalentlylinked to an intracellular carrier peptide, wherein the intracellularcarrier peptide, the modulatory peptide, or both are modified at theN-terminus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph plotting CK release against concentrations oftherapeutic peptides KAI-9706 and KAI-1455.

FIG. 2 shows a graph plotting percent infarction against increasingconcentrations of therapeutic peptides KAI-9706 and KAI-1455.

FIGS. 3A-3C show graphs plotting percent of intact therapeutic peptidesKAI-9803, KAI-9706, and KAI-1455, surviving over time in human serum(FIG. 3A), pig serum (FIG. 3B), and rat serum (FIG. 3C).

FIGS. 4A-4B show the conversion of a therapeutic peptide comprising acarrier peptide and a cargo peptide linked via a disulfide bond (FIG.4A) to a therapeutic peptide in linear form (FIG. 4B). This linearpeptide (KP-01547; FIG. 4B) has been capped at its amino and carboxytermini and contains a short amino acid sequence linker.

FIG. 5 shows a graph plotting percent of intact therapeutic peptides(linear and non-linear) over time (days).

FIG. 6 shows a graph comparing the stability of therapeutic peptidesover time (days).

FIG. 7 shows two linear peptides.

FIG. 8 shows a graph comparing the stability of various PKC-β_(I)therapeutic peptides over time.

FIG. 9 shows a graph a graph comparing the stability of variousPKC-β_(II) therapeutic peptides over time.

FIGS. 10A-10D show graphs illustrating the impact of temperature on thestability KAI-9706 at 26° C. (FIG. 10A) and 37° C. (FIG. 10C), and onKAI 1455 at 26° C. (FIG. 10B) and 37° C. (FIG. 10D).

FIG. 11 shows a graph plotting creatine kinase release in the presenceof increasing concentrations of KAI-9803 or KAI-1355 peptides in aLangendorff in vitro post-ischemia assay, with 10 minutes perfusion.

FIG. 12 shows an illustration depicting the construction of KAI-1479.

FIGS. 13A and 13B show bar graphs of results from a reperfusion study,wherein 13A illustrates the time line of events during the experimentand 13B shows a bar graph illustrating the protective properties ofvarious therapeutic peptides (KAI-9803, KAI-1479, and KAI-1482).

FIG. 14 shows a illustrates the cargo peptide of SEQ ID NO:33 with acysteine residue at the amino terminus of the peptide.

FIG. 15A-15D show four different possible configurations of therapeuticpeptide multimers.

FIG. 16 shows linear peptides KP-1680, KP-1681, KP-1633, and KP-1678.

FIG. 17A-17D show graphs of the percent of peptides remaining in testsolutions over time. FIG. 17A shows the percent of peptides remaining intest solutions over time. FIG. 17B shows the percent of peptidesremaining in test solutions over time. FIG. 17C shows the percent ofpeptides remaining in test solutions over time. FIG. 17D shows thepercent of peptides remaining in test solutions over time.

MODES OF CARRYING OUT THE INVENTION

The disclosed invention relates to methods of modifying peptidecompositions to increase stability and delivery efficiency.Specifically, the disclosed invention relates to methods to increase thestability and delivery efficiency of protein kinase C (PKC) modulatorypeptide compositions. A “therapeutic peptide composition” comprises a“carrier peptide” and a “cargo peptide.” A “carrier peptide” is apeptide or amino acid sequence within a peptide that facilitates thecellular uptake of the therapeutic peptide composition. The “cargopeptide” is a PKC modulatory peptide. Peptide modifications to eitherthe carrier peptide, the cargo peptide, or both, which are describedherein increase the stability and delivery efficiency of therapeuticpeptide compositions by reducing disulfide bond exchange, physicalstability, reducing proteolytic degradation, and increasing efficiencyof cellular uptake.

Disulfide Bond Exchange

A preferred embodiment of the disclosed therapeutic peptide compositionsprovides a cargo peptide coupled to a carrier peptide via a disulfidebond between two joining sulfur-containing residues, one in eachpeptide. The disulfide bond of this embodiment can be unstable whetherthe therapeutic peptide composition is in solution, lyophilized,precipitated, crystallized, or spray-dried, leading to carrier-cargocombinations to degrade to carrier-carrier compositions, which areinactive, and cargo-cargo compositions, which are also inactive and arefrequently insoluble. The stability of the disclosed therapeutic peptidecompositions is improved through the use of chemical modifications andby controlling the physical environment of the peptide compositionsprior to use.

Chemical Modifications

The joining sulfur-containing residue can be placed anywhere in thesequence of the carrier or cargo peptides. For example, a preferredembodiment of the disclosed therapeutic peptide composition typicallyhas the joining sulfur-containing residue at the amino terminus of thecarrier and cargo peptides. The joining sulfur-containing residues canbe placed at the carboxy termini of the peptides, or alternatively atthe amino terminus of peptide and at the carboxy terminus of the otherpeptide. Additionally, the joining sulfur-containing residue can beplaced anywhere within the sequence of either or both of the peptides.Placing the joining sulfur-containing residue within the carrierpeptide, the cargo peptide, or both has been observed to reduce the rateof disulfide bond exchange.

An example of chemical modifications useful to stabilize the disulfidebonds of the therapeutic peptide compositions involves optimizing theamino acid residue or residues immediately proximate to thesulfur-containing residues used to join the carrier and cargo peptide. Apreferred method of stabilizing the disulfide bond involves placing analiphatic residue immediately proximate to the sulfur-containing residuein the carrier and/or cargo peptides. Aliphatic residues includealanine, valine, leucine and isoleucine. Thus, when the joiningsulfur-containing residue is placed at the amino terminus of a peptide,an aliphatic residue is placed at the penultimate amino terminalposition of the peptide to reduce the rate of disulfide bond exchange.When the joining sulfur-containing residue is located at the carboxyterminus of a peptide, an aliphatic residue is placed at the penultimatecarboxy terminal position of the peptide to reduce the rate of disulfidebond exchange. When the joining sulfur-containing residue is locatedwithin the sequence of a peptide, the aliphatic residue can be place ateither the amino terminal or carboxy terminal side of the residue, or atboth sides.

A variety of sulfur-containing residues are contemplated for use withthe presently disclosed invention. Cysteine and cysteine analogs canalso be used as the joining cysteine residues in the peptidecomposition. Particular cysteine analogs include D-cysteine,homocysteine, alpha-methyl cysteine, mercaptopropionic acid,mercaptoacetic acid, penicillamine, acetylated forms of those analogscapable of accepting an acetyl group, and cysteine analogs modified withother blocking groups. For example, the use of homocysteine, acetylatedhomocysteine, penicillamine, and acetylated penicillamine in the cargo,the carrier, or both peptides have been shown to stabilize the peptidecomposition and decrease disulfide bond exchange. Alpha-methyl cysteineinhibits disulfide degration because the base-mediated abstraction ofthe alpha hydrogen from one cysteine is prevented by the presence of thesulfur atom. Cargo/carrier peptide conjugates joined by disulfide bondshave been shown to be more resistant to glutathione reduction thanunmodified peptides. Other cysteine analogs are also useful as joiningcysteines. Similarly, stereoisomers of cysteine will inhibit disulfidebond exchange.

Disulfide bond exchange can be eliminated completely by linking thecarrier and cargo peptides to form a single, linear peptide. This methodis discussed below.

Physical Stability

The physical environment of the disulfide has an effect on stability. Asshown (in part) in FIGS. 10A, 10B, 10C and 10D, stability increases insolution as the pH of the solution decreases (acidic environment betterthan basic), the temperature of the solution decreases, and as theconcentration of the peptide composition in solution decreases. In thelyophilized form, stability increases as the pH decreases, thetemperature decreases, and the ratio of the peptide composition toexcipient increases. Preferred excipients are discussed in U.S. patentapplication Ser. No. 11/240,962, filed Sep. 30, 2005, which is herebyincorporated by reference in its entirety.

The unexpected “excipient effect” was most pronounced for mannitol,which is a highly crystalline excipient. Using less crystallineexcipients (such as sucrose) or even using no excipient, showed muchless dependency on peptide composition quantity. Although not wishing tobe bound or limited by any theory, it is thought that use of anon-crystalline excipient creates an amorphous matrix, which helpsprevent intermolecular associations. Theoretically, in a crystallinematrix the peptide composition is excluded and forced to the walls ofthe vial, perhaps causing high local concentrations. With low amount ofAPI the resulting thin film has high peptide-glass contact area and thesilica is destabilizing.

A number of factors impact the efficiency with which a therapeuticpeptide composition is taken up by a target cell. For example, thesolubility of a therapeutic peptide impacts the efficiency with whichthe peptide is taken up by a target cell. In turn, the amino acidsequence of a carrier or cargo peptide largely determines thatsolubility the peptide compositions in which they are used. Somepeptides, particularly cargo peptides, will contain hydrophobicresidues, (e.g., Phe, Tyr, Leu), with regular spacing which allows forintramolecular interactions by a “zipper” mechanism leading toaggregation. An example of such a potentially problematic peptide isshown in FIG. 14. The illustrated sequence is believed to form abeta-strand in the VI domain of δPKC. Such peptides have the tendency toform insoluble deposits.

The solubility of such peptides can be improved by making certainmodifications to the cargo peptide sequence. For example, theintroduction of solubilizing groups at amino and or carboxy termini oron internal residues, such as hydrating groups, like polyethylene glycol(PEG), highly charged groups, like quaternary ammonium salts, or bulky,branched chains of particular amino acid residues will improve thesolubility of peptides like the one illustrated in FIG. 14.Additionally, those hydrophobic side chains that are shown not to berequired for activity can be eliminated by deletion or substitution witha conservative or non-interfering residue, such as an alanine, glycine,or serine, thus improving the solubility of the peptides.

Proteolytic Degradation: Plasma Stability

Blood and plasma contain proteases which can degrade the protein kinaseC modulatory peptides disclosed herein or the carrier peptides whichfacilitate the cellular uptake of the peptide composition, or both. Onemethod to decrease proteolytic degradation of the carrier or cargopeptides is to mask the targets of the proteases presented by thetherapeutic peptide composition.

Once the therapeutic peptide enters the plasma of a subject, it becomevulnerable to attack by peptidases. Strategies are provided whichaddress peptide degradation caused by exopeptidases (any of a group ofenzymes that hydrolyze peptide bonds formed by the terminal amino acidsof peptide chains) or endopeptidases (any of a group of enzymes thathydrolyze peptide bonds within the long chains of protein molecules).Exopeptidases are enzymes that cleave amino acid residues from the aminoor carboxy termini of a peptide or protein, and can cleave at specificor non-specific sites. Endopeptidases, which cleave within an amino acidsequence, can also be non-specific, however endopeptidases frequentlyrecognize particular amino sequences (recognition sites) and cleaves thepeptide at or near those sites.

One method of protecting peptide compositions from proteolyticdegradation involves the “capping” the amino and/or carboxy termini ofthe peptides. The term “capping” refers to the introduction of ablocking group to the terminus of the peptide via a covalentmodification. Suitable blocking groups serve to cap the termini of thepeptides without decreasing the biological activity of the peptides.Acetylation of the amino termini of the described peptides is apreferred method of protecting the peptides from proteolyticdegradation. Other capping moieties are possible. The selection ofacylating moiety provides an opportunity to “cap” the peptide as well asadjust the hydrophobicity of the compound. For example, thehydrophobicity increases for the following acyl group series: formyl,acetyl, propanoyl, hexanoyl, myristoyl, and are also contemplated ascapping moieties. Amidation of the carboxy termini of the describedpeptides is also a preferred method of protecting the peptides fromproteolytic degradation.

Protecting peptides from endopeptidases typically involvesidentification and elimination of an endopeptidase recognition site froma peptide. Protease recognition cites are well known to those ofordinary skill in the art. Thus it is possible to identity a potentialendoprotease recognition site and then eliminating that site by alteringthe amino acid sequence within the recognition site. Residues in therecognition sequence can be moved or removed to destroy the recognitionsite. Preferably, a conservative substitution is made with one or moreof the amino acids which comprise an identified protease recognitionsite. The side chains of these amino acids possess a variety of chemicalproperties. For the purposes of the present discussion, the most commonamino acids are categorized into 9 groups, listed below. Substitutionwithin these groups is considered to be a conservative substitution.

Conservative Amino Acid Substitution Small/Aliphatic residues: Gly, Ala,Val, Leu, Ile Cyclic Imino Acid: Pro Hydroxyl Residues: Ser, Thr AcidicResidues: Asp, Glu Amide Residues: Asn, Gln Basic Residues: Lys, ArgImidazole Residue: His Aromatic Residues: Phe, Tyr, TrpSulfur-Containing Residues: Met, Cys

Efficiency of Cellular Uptake

In addition to the modifications discussed above, improve utility forthe disclosed therapeutic peptide compositions can be achieved byaltering the linkage of the carrier and cargo peptides. For example, inone embodiment, carrier and cargo peptides are linked by a peptide bondto form a linear peptide. Stability and potency of the therapeuticpeptides can also be increased through the construction of peptidemultimers, wherein a plurality of cargo peptides is linked to one ormore carrier peptides. An additional embodiment of the inventioninvolving a cleavable linker sequence is also discussed.

Linear Peptides

Another strategy to improve peptide composition stability involvesjoining the cargo and carrier peptides into a single peptide as opposedto joining the peptides via a disulfide bond. For example, in theembodiment shown in FIG. 4A, the cargo peptide (SEQ ID NO:13) linked viaamino terminal cysteines. A linear version of the cargo and carrierpeptides is shown in FIG. 4B, where the cargo and carrier peptides arelinked via a short dipeptide linker (e.g., Ser-Gly). This linker isexemplary.

In the example illustrated, the C-terminus of cargo is linked to theN-terminus of the carrier via the linker. However, the other possiblepermutations are also contemplated, including linking the peptide viathere C-termini, their N-termini, and where the carrier peptide islocated at the N-terminal portion of the peptide composition.

Additionally, the steps discussed above to stabilize a disulfide bondlinked peptide composition can also be used with a linear, whereappropriate. For example, the linear peptide composition shown in FIG.4B has been capped at both its amino and carboxy termini. Moreoversequences within the peptide can be scrambled or substituted withD-amino acids.

As shown in FIG. 7, deamination of Asn at position 7 of the β-I had beenobserved to cause significant instability in the linearized version ofthe peptide composition linked by Asn-Gly. Changing the Gly to Leustabilized this linear peptide composition. Similarly, deamination ofthe Gln residue at position 2 of the linear β-II composition wasobserved to cause significant instability. Substitution with Gluimproved stability of the linear composition. Data comparing themodified versions of these peptides is shown in FIGS. 8 and 9.

Without being limited to any particular theory, it is thought thatdeamination results from the attack of the alpha or main-chain amideHN—C-terminal to the Asn residue on the side-chain amide of Asn,generating the cyclic aspartamide intermediate which can hydrolyze to anaspartic acid moiety. Increasing the size of the residue C-terminal toAsn is thought to increase the steric hinderance on the main-chainamide, significantly slowing deamidation.

Peptide Multimers

Another method of improving stability and potency is available byforming multimers with a plurality of cargo peptides associated with oneor more carrier peptides. Examples of such formulations are shown inFIG. 15. Branched, multivalent peptide compositions will increaseavidity, potency and stability of the compositions. By engineeringcleavage sites or other release mechanisms into the multimercompositions, the multiple conjugates can release nearly simultaneously,PKC modulatory cargo peptides inside a target cell. An example ofmultimeric peptides is discussed in Yu et al. JBC 275(6):3943-9 (2000).

Cleavable Sequence

Typically the carrier and cargo are linked by a linkage that can becleaved by ubiquitous enzymes such as esterases, amidases, and the like.It is assumed that the concentration of such enzymes is higher insidecells rather than in the extracellular milieu. Thus, once the conjugateis inside a cell, it is more likely to encounter an enzyme that cancleave the linkage between cargo and carrier. The enzyme can thusrelease the biologically active cargo inside a cell, where it presumablyis most useful.

Protein Kinase C Modulatory Peptides

The term protein kinase C modulatory peptide refers to a peptide derivedfrom a PKC isozyme- and/or variable region. Various PKC isozyme- andvariable region-specific peptides have been described and can be usedwith the presently disclosed invention. Preferably, the PKC modulatorypeptide is a V1, V3 or VS-derived peptide. (The terminology “V1” and“C2” are synonymous.) The following US patents or patent applicationsdescribe a variety of suitable peptides that can be used with thepresently disclosed invention: U.S. Pat. Nos. 5,783,405, 6,165,977,6,855,693, US2004/0204364, US2002/0150984, US2002/0168354,US2002/057413, US2003/0223981, US2004/0009922 and Ser. No. 10/428,280,each of which are incorporated herein by reference in their entirety.Table 1 provides a listing of preferred PKC modulatory peptides for usewith the present invention.

TABLE 1 Cargo Peptides derived from PKC isozymes Peptide SEQ ID NO.Sequence αV3-1 SEQ ID NO: 2 I-P-E-G-D-E-E-G αV5-1 SEQ ID NO: 3Q-L-V-I-A-N αV5-1.1 SEQ ID NO: 4 G-L-G-A-E-N αV5-1.2 SEQ ID NO: 5A-R-G-A-E-N αV5-1.3 SEQ ID NO: 6 C-G-K-G-A-E-N αV5-1.4 SEQ ID NO: 7C-G-K-G-A-E-N βC2-1 SEQ ID NO: 8 K-Q-K-T-K-T-I-K βC2-2 SEQ ID NO: 9M-D-P-N-G-L-S-D-P-Y-V-K-L βC2-3 SEQ ID NO: 10 I-P-D-P-K-S-E βC2-4SEQ ID NO: 11 S-L-N-P-E-W-N-E-T βV3-1 SEQ ID NO: 12 V-P-P-E-G-S-E-AβIV5-1 SEQ ID NO: 13 K-L-F-I-M-N βIV5-2 SEQ ID NO: 14 R-D-K-R-D-T-SβIV5-2.1 SEQ ID NO: 15 C-A-R-D-K-R-D-T-S βIV5-2.2 SEQ ID NO: 16G-R-D-K-R-D-T-S βIV5-2.3 SEQ ID NO: 17 A-R-D-K-R-D-T-S βIV5-3SEQ ID NO: 18 A-R-D-K-R-D-T-S-N-F-D-K βIV5-4 SEQ ID NO: 19A-G-F-S-Y-T-N-P-E-F-V-I-N-V βIIV5-1 SEQ ID NO: 20 Q-E-V-I-R-N βIIV5-2SEQ ID NO: 21 C-G-R-N-A-E βIIV5-3 SEQ ID NO: 22 A-C-G-R-N-A-E βIIV5-3.1SEQ ID NO: 23 A-C-G-K-N-A-E βIIV5-4 SEQ ID NO: 24 K-A-C-G-R-N-A-EβIIV5-5 SEQ ID NO: 25 C-G-R-N-A-E-N βIIV5-6 SEQ ID NO: 26 A-C-G-R-N-A-EβIIV5-7 SEQ ID NO: 27 S-F-V-N-S-E-F-L-K-P-E-V-L-S γV3-1 SEQ ID NO: 28V-A-D-A-D-N-C-S γV5-1 SEQ ID NO: 29 G-R-S-G-E-N γV5-1.1 SEQ ID NO: 30G-L-S-G-E-N γV5-2 SEQ ID NO: 31 R-L-V-L-A-S γV5-3 SEQ ID NO: 32P-C-G-R-S-G-E-N δV1-1 SEQ ID NO: 33 C-S-F-N-S-Y-E-L-G-S-L 1eu-TruncateSEQ ID NO: 165 C-S-F-N-S-Y-E-L-G-S δV1-1.1 SEQ ID NO: 34S-F-N-S-Y-E-L-G-S-L δV1-1.2 SEQ ID NO: 35 T-F-N-S-Y-E-L-G-S-L δV1-1.3SEQ ID NO: 36 A-F-N-S-N-Y-E-L-G-S-L δV1-1.4 SEQ ID NO: 37S-F-N-S-Y-E-L-G-T-L δV1-1.5 SEQ ID NO: 38 S-T-N-S-Y-E-L-G-S-L δV1-1.6SEQ ID NO: 39 S-F-N-S-F-E-L-G-S-L δV1-1.7 SEQ ID NO: 40S-N-S-Y-D-L-G-S-L δV1-1.8 SEQ ID NO: 41 S-F-N-S-Y-E-L-P-S-L δV1-1.9SEQ ID NO: 42 T-F-N-S-Y-E-L-G-T-L δV1-1.10 SEQ ID NO: 43S-F-N-S-Y-E-I-G-S-V δV1-1.11 SEQ ID NO: 44 S-F-N-S-Y-E-V-G-S-I δV1-1.12SEQ ID NO: 45 S-F-N-S-Y-E-L-G-S-V δV1-1.13 SEQ ID NO: 46S-F-N-S-Y-E-L-G-S-I δV1-1.14 SEQ ID NO: 47 S-F-N-S-Y-E-I-G-S-L δV1-1.15SEQ ID NO: 48 S-F-N-S-Y-E-V-G-S-L δV1-1.16 SEQ ID NO: 49A-F-N-S-Y-E-L-G-S-L δV1-1.17 SEQ ID NO: 50 Y-D-L-G-S-L δV1-1.18SEQ ID NO: 51 F-D-L-G-S-L δV1-1.19 SEQ ID NO: 52 Y-D-I-G-S-L δV1-1.20SEQ ID NO: 53 Y-D-V-G-S-L δV1-1.21 SEQ ID NO: 54 Y-D-L-P-S-L δV1-1.22SEQ ID NO: 55 Y-D-L-G-L-L δV1-1.23 SEQ ID NO: 56 Y-D-L-G-S-I δV1-1.24SEQ ID NO: 57 Y-D-L-G-S-V δV1-1.25 SEQ ID NO: 58 I-G-S-L δV1-1.26SEQ ID NO: 59 V-G-S-L δV1-1.27 SEQ ID NO: 60 L-P-S-L δV1-1.28SEQ ID NO: 61 L-G-L-L δV1-1.29 SEQ ID NO: 62 L-G-S-I δV1-1.30SEQ ID NO: 63 L-G-S-V δV1-2 SEQ ID NO: 64 A-L-S-T-E-R-G-K-T-L-V δV1-2.1SEQ ID NO: 65 A-L-S-T-D-R-G-K-T-L-V δV1-2.2 SEQ ID NO: 66A-L-T-S-D-R-G-K-T-L-V δV1-2.3 SEQ ID NO: 67 A-L-T-T-D-R-G-K-S-L-VδV1-2.4 SEQ ID NO: 68 A-L-T-T-D-R-P-K-T-L-V δV1-2.5 SEQ ID NO: 69A-L-T-T-D-R-G-R-T-L-V δV1-2.6 SEQ ID NO: 70 A-L-T-T-D-K-G-K-T-L-VδV1-2.7 SEQ ID NO: 71 A-L-T-T-D-K-G-K-T-L-V δV1-3 SEQ ID NO: 72V-L-M-R-A-A-E-E-P-V δV1-4 SEQ ID NO: 73 Q-S-M-R-S-E-D-E-A-K δV1-5SEQ ID NO: 163 A-F-N-S-Y-E-L-G-S δv3-1 SEQ ID NO: 74 Q-G-F-E-K-K-T-G-Vδv3-2 SEQ ID NO: 75 D-N-N-G-T-Y-G-K-I δv5-1 SEQ ID NO: 76 K-N-L-I-D-Sδv5-2 SEQ ID NO: 77 V-K-S-P-R-D-Y-S δv5-2.1 SEQ ID NO: 78V-K-S-P-C-R-D-Y-S δv5-2.2 SEQ ID NO: 79 I-K-S-P-R-L-Y-S δv5-3SEQ ID NO: 80 K-N-L-I-D-S δv5-4 SEQ ID NO: 81 P-K-V-K-S-P-R-D-Y-S-NϵV1-1 SEQ ID NO: 82 N-G-L-L-K-I-K ϵV1-2 SEQ ID NO: 83 E-A-V-S-L-K-P-TϵV1-3 SEQ ID NO: 84 L-A-V-F-H-D-A-P-I-G-Y ϵV1-4 SEQ ID NO: 85D-D-F-V-A-N-C-T-I ϵV1-5 SEQ ID NO: 86 W-I-D-L-E-P-E-G-R-V ϵV1-6SEQ ID NO: 87 H-A-V-G-P-R-P-Q-T-F ϵV1-7 SEQ ID NO: 88 N-G-S-R-H-F-E-DϵV1-7.1 SEQ ID NO: 89 H-D-A-P-I-G-Y-D ϵV1-7.2 SEQ ID NO: 90 H-D-A-P-I-GϵV1-7.3 SEQ ID NO: 91 H-D-A-A-I-G-Y-D ϵV1-7.4 SEQ ID NO: 92H-D-A-P-I-P-Y-D ϵV1-7.5 SEQ ID NO: 93 H-N-A-P-I-G-Y-D ϵV1-7.6SEQ ID NO: 94 H-A-A-P-I-G-Y-D ϵV1-7.7 SEQ ID NO: 95 A-D-A-P-I-G-Y-DϵV1-7.8 SEQ ID NO: 96 H-D-A-P-A-G-Y-D ϵV1-7.9 SEQ ID NO: 97H-D-A-P-I-G-A-D ϵV1-7.10 SEQ ID NO: 98 H-D-A-P-I-A-Y-D ϵV1-7.11SEQ ID NO: 99 H-D-A-P-I-G-Y-A ϵV3-1 SEQ ID NO: 100 S-S-P-S-E-E-D-R-SϵV3-2 SEQ ID NO: 101 P-C-D-Q-E-I-K-E ϵV3-3 SEQ ID NO: 102E-N-N-I-R-K-A-L-S ϵV3-4 SEQ ID NO: 103 G-E-V-R-Q-G-Q-A ϵV5-1SEQ ID NO: 104 E-A-V-K-Q ϵV5-2 SEQ ID NO: 105 I-K-T-K-R-D-V ϵV5-2.1SEQ ID NO: 106 I-K-T-K-R-L-I ϵV5-3 SEQ ID NO: 107 C-E-A-I-V-K-Q ϵV5-4SEQ ID NO: 108 T-K-R-D-V-N-N-F-D-Q ζv1-1 SEQ ID NO: 109 V-R-L-K-A-H-Yζv1-2 SEQ ID NO: 110 V-D-S-E-G-D ζv1-3 SEQ ID NO: 111 V-F-P-S-I-P-E-Qζv3-1 SEQ ID NO: 112 S-Q-E-P-P-V-D-D-K-N-E-D-A-D-L ζv3-2 SEQ ID NO: 113I-K-D-D-S-E-D ζv3-3 SEQ ID NO: 114 P-V-I-D-G-M-D-G-I ζv5-1SEQ ID NO: 115 E-D-A-I-K-R ζv5-1.1 SEQ ID NO: 116 E-D-A-I-R ζv5-2SEQ ID NO: 117 I-T-D-D-Y-G-L-D ζv5-2.1 SEQ ID NO: 118 I-T-D-D-Y-G-D-Lζv5-3 SEQ ID NO: 119 D-D-Y-G-L-D-N ηV1-1 SEQ ID NO: 120 N-G-Y-L-R-V-RηV1-2 SEQ ID NO: 121 E-A-V-G-L-Q-P-T ηV1-3 SEQ ID NO: 122L-A-V-F-H-E-T-P-L-G-Y ηV1-4 SEQ ID NO: 123 D-F-V-A-N-C-T-L ηV1-5SEQ ID NO: 124 W-V-D-L-E-P-E-G-K-V ηV1-6 SEQ ID NO: 125 H-S-L-F-K-K-G-HηV1-7 SEQ ID NO: 126 T-G-A-S-D-T-F-E-G ηV5-1 SEQ ID NO: 127 E-G-H-L-P-MηV5-1.1 SEQ ID NO: 128 E-G-H-D-P-M ηV5-2 SEQ ID NO: 129 I-K-S-R-E-D-V-SηV5-3 SEQ ID NO: 130 V-R-S-R-E-D-V-S ηV5-4 SEQ ID NO: 131P-R-I-K-S-R-E-D-V λV1-1 SEQ ID NO: 132 H-Q-V-R-V-K-A-Y-Y-R λV1-2SEQ ID NO: 133 Y-E-L-N-K-D-S-E-L-L-I λV3-1 SEQ ID NO: 134M-D-Q-S-S-M-H-S-D-H-A-Q-T-V-I λV3-2 SEQ ID NO: 135 L-D-Q-V-G-E-E λV3-3SEQ ID NO: 136 E-A-M-N-T-R-E-S-G λV5-1 SEQ ID NO: 137 D-D-I-V-R-K μV5-2SEQ ID NO: 138 V-K-L-C-D-F-G-F μV5-2.1 SEQ ID NO: 139 I-R-L-C-D-F-A-FμV5-3 SEQ ID NO: 140 Q-V-K-L-C-D-F-G-F-A μV1-1 SEQ ID NO: 141M-S-V-P-P-L-L-R-P μV1-2 SEQ ID NO: 142 K-F-P-E-C-G-F-Y-G-L-Y μV3-1SEQ ID NO: 143 D-P-D-A-D-Q-E-D-S μV3-2 SEQ ID NO: 144 S-K-D-T-L-R-K-R-HμV3-3 SEQ ID NO: 145 I-T-L-F-Q-N-D-T-G μV3-4 SEQ ID NO: 146G-S-N-S-H-K-D-I-S μV5-1 SEQ ID NO: 147 S-D-S-P-E-A θV1-1 SEQ ID NO: 148G-L-S-N-F-D-C-G θV1-2 SEQ ID NO: 149 Y-V-E-S-E-N-G-Q-M-Y-I θV1-3SEQ ID NO: 150 I-V-K-G-K-N-V-D-L-I θV1-4 SEQ ID NO: 151D-M-N-E-F-E-T-E-G-F θV3-1 SEQ ID NO: 152 C-S-I-K-N-E-A-R-L θV3-2SEQ ID NO: 153 G-K-R-E-P-Q-G-I-S θV3-3 SEQ ID NO: 154 D-E-V-D-K-M-C-H-LθV5-1 SEQ ID NO: 155 R-A-L-I-N-S θV5-2 SEQ ID NO: 156 V-K-S-P-F-D-C-SθV5-2.1 SEQ ID NO: 157 V-R-S-P-F-D-C-S θV5-3 SEQ ID NO: 158D-R-A-L-I-N-S ιV5-1 SEQ ID NO: 159 I-S-G-E-F-G-L-D ιV5-1.1SEQ ID NO: 160 C-S-G-E-F-G-L-D ιV5-2 SEQ ID NO: 161 D-D-D-I-V-R-K ιV5-3SEQ ID NO: 162 D-D-I-V-R-K

TABLE 2 Carrier Peptides TAT Carrier SEQ ID NO: 166 YGRKKRRQRRR PeptideTAT Carrier SEQ ID NO: 164 CYGRKKRRQRRR Peptide with N- terminal Cys

Other examples of carries include octa-Arg, octa-D-Arg, and Antennapediaderived peptides, which are known in the art.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Exopeptidase Protection: Plasma Stability of Capped Peptides

Plasma stability of capped peptides was compared. KAI-9706 was modifiedstepwise at its amino and carboxy termini. Plasma stability as measuredby the percent of peptide composition remaining after 15 minutes. Theresults are provided in Table 2.

TABLE 2 Plasma Stability of KAI-9706 cargo H—OH Ac—OH H—NH₂ Ac—NH₂carrier H—OH 1 11  0 0 Ac—OH 57 nd nd 48 H—NH₂ 60 nd nd 51 Ac—NH₂ 92 9390 90 % parent remaining at 15 mins t_(1/2) in rat plasma = 40-45 minsfor longest-lived derivatives

The data provided above shows that the peptide composition, comprisingunmodified cargo and carrier peptides, was the least stable. Moreover,protection of the carrier peptide alone failed to increase the half lifeof the peptide composition in plasma. Moreover, modification of thecargo peptide with the carrier peptide unmodified had no apparent effecton half-life stability in plasma. However, the addition of protectinggroups to the carrier peptide, whether at the amino or carboxy terminilead to a marked and nearly equivalent increase in plasma stability forthe peptide composition. Protection of both groups in the carrierpeptide provided additional protection. Interestingly, protection of thecargo peptide had little or no effect on the stability of thecomposition.

Example 2 Effect of D-Peptides on Plasma Stability

KAI-9706 was engineered with D-amino acids to determine their impact onpeptide composition stability. Unmodified KAI-9706 was compared to apeptide composition with the same amino acid sequence, however the aminoacids used were d-enantiomers instead of 1-amino acids. A retro-inversoversion and a scrambled version of the peptide composition were alsoprepared. The data from the experiment is shown in Table 3.

TABLE 3 Plasma Stability of KAI-9706 cargo All-L All-D scrambled R/Icarrier All-L 1 0 2 All-D 88 100 67 R/I 100 % parent remaining at 15mins

Modification of the carrier showed the great propensity in improving thehalf life of the composition while modification of the cargo showedlittle effect.

Example 3 Capped KAI-9706 Maintains In Vitro Potency

Capping the carrier peptide portion KAI-9706 (KAI-1455) was shown toincrease the plasma half life of the peptide composition. The ability ofthe capped composition to inhibit ischemic damage in a rat heat model(Langendorff Assay) was evaluated versus the uncapped form. The resultsare shown in FIG. 1.

Example 4 Capped KAI-9706 Shows Increased Potency

KAI-1455 was tested in a stroke model. As shown in FIG. 2, the cappedversion of the peptide composition provided increased protection tobrain tissue as judged by percent infract. This data shows thatsignificant protection of brain tissue was achieved at lower doses.

Example 5 Peptide Stability is Increased Regardless of Species

The stability of modified KAI-9706 peptide (KAI-1455) was comparedagainst KAI-9706 and KAI-9803 in human (FIG. 3A), pig (FIG. 3B) and ratserum (FIG. 3C). The capped version, KAI-1455, showed increased plasmastability in all three species.

Example 6 Capped KAI-9706 Shows Increased Potency

KAI-1455 was tested in a stroke model. As shown in FIG. 2, the cappedversion of the peptide composition provided increased protection tobrain tissue as judged by percent infract. This data shows thatsignificant protection of brain tissue was achieved at lower doses.

Example 7 Peptide Stability is Increased Regardless of Species

The stability of modified KAI-9706 peptide (KAI-1455) was comparedagainst KAI-9706 and KAI-9803 in human (FIG. 3A), pig (FIG. 3B) and ratserum (FIG. 3C). The capped version, KAI-1455, showed increased plasmastability in all three species.

Example 8 Stability of Linear Peptides

Linear versions of KAI-9803 and BC2-4 were constructed to evaluate theirstability relative to disulfide bond linked versions of these and otherpeptide compositions. The peptides were placed in solution at 0.1 mg/mlin PBS (pH 7.4) at 37° C. As shown in FIG. 5, the linear versions ofKAI-9803 and BV2-4 showed increased stability.

Example 9 Linear PKC-β_(I) and PKC-β_(II) Peptide Compositions ShowIncreased Stability Over Disulfide Linked Compositions

Linear and disulfide bond linked versions of PKC-β_(I) and PKC-β_(II)peptide compositions were incubated under the conditions described inExample 8. As can be seen in FIG. 6, the linear forms of the PKC-β_(I)and PKC-β_(II) peptides showed increased stability as compared to theirnon-linear counterparts.

Example 10 Improved Stability of Linear PKC-β_(I) and PKC-β_(II) PeptideCompositions

The linear versions of PKC-β_(I) and PKC-β_(II) peptide compositionsshowed improved stability but were also the subject of deaminationreactions. In particular, the Asn residues at position 7 of the PKC-βIand PKC-βII peptides and the Gln at position 2 of the PKC-βII peptide.These linear peptide compositions were modified by substituting the Glyimmediately C-terminal to the Asn with either Leu in the βI peptidecomposition or Gly to Ile in the βII peptide composition. The Gln atposition 2 of the βII peptide composition was also substituted with aGlu residue. The stability of the peptides was studied under theconditions described in Example 8. As shown in FIGS. 8 and 9, the aminoacid substitutions discussed above served to stabilize these linearpeptide compositions.

Example 11 KAI-9803 Derivative (KAI-1355) Maintains Potency

A truncated version of KAI-9803, KAI-1355, in which the carboxy terminalleucine was removed was tested for potency. Stability studies withKAI-1355 showed that deletion of the C-terminal Leu residue increasedthe stability of this cargo peptide. Potency of the derivative peptidecomposition was compared to that of the full length version, KAI-9803 ina Langendorff in vitro post-ischemia model. The results of theexperiment are shown in FIG. 11. As shown, KAI-1355 (the modifiedversion of KAI-9803) is still capable of protecting cardiac tissue fromischemia with potency comparable to that of the full length KAI-9803.

Example 12 Optimization of KAI-9803 to Produce KAI-1479

Having demonstrated that truncation of the cargo peptide of KAI-9803 hadlittle or now effect on potency, while stabilizing the peptidecomposition. As illustrated in FIG. 12, a capped version of the TATcarrier peptide was bound to the truncated cargo peptide of KAI-1355,producing KAI-1479, which comprises a truncated 9803 cargo peptide and afully capped TAT carrier peptide.

The modified KAI-1479, KAI-9803 and KAI-1482 peptide compositions wereassayed in a rat middle cerebral artery occlusion (MCAO) stroke model todetermine the ability of the peptide compositions to inhibit infarctsize. The rats were subjected to a 2 hour period of cerebral arterialocclusion. Each of the peptide compositions or saline was administeredto the test animals immediately prior to a 22 hour reperfusion period,after which time the animals were sacrificed and the infarct size wasmeasured. As shown in FIG. 13, the modified KAI-1479 peptide compositionshowed an increased ability to retard infarct size as compared toKAI-9803. KP-01482 has a cargo sequence CELGSLQAEDD (SEQ ID NO: 1)linked to a TAT peptide with a N-Term Cys, which is capped at both endsand disulfide conjugated to the cargo.

Example 13 In Vitro Biological Stability of a Series of Linear EpsilonPKC Inhibitors

The effect of N-terminal acetylation and C-terminal amidation oncompound stability in plasma and serum from rat and human was studied.The linear peptides examined are shown in FIG. 16. The compounds weretested in the plasma/serum at a concentration of 100 μg/ml. Thesolutions were incubated at room temperature, precipitated with 5% TCA,and then the supernatant was neutralized with ammonium acetate. Thepeptides were then analyzed by LC/MS. As can be seen from the data inFIGS. 17A-17D, all the tested compounds were relatively stable in humanplasma but KP-1633 and KP-1678, containing C-terminal amides, showedincreased stability in human serum. N-terminal acetylation alone did notstabilize the peptides. Interestingly, the amino acid sequence ofKP-1680 and its degradation products indicated that the metabolizedforms of the peptide showed sequential cleavage of arginine residuesfrom the C-terminus. Carboxypeptidase N activity in the serum but notthe plasma could account for the difference in observed stability. Theplasma samples were collected with EDTA, which is known to inhibit thiszinc metalloprotease.

I claim:
 1. A composition comprising a protein kinase C (PKC) modulatorypeptide conjugate and a pharmaceutically acceptable excipient, whereinthe conjugate comprises a PKC modulatory peptide having a first cysteineanalog covalently linked by a disulfide bond to an intracellular carrierpeptide having a second cysteine analog, wherein the intracellularcarrier peptide is CYGRKKRRQRRR (SEQ ID NO: 164) and is substituted atits N-terminal end by an acyl, alkyl, or sulfonyl group, and wherein thefirst and second cysteine analogs are independently selected frompenicillamine, alpha-methyl cysteine, and acetylated forms thereof. 2.The composition of claim 1, wherein the PKC modulatory peptide is aninhibitory peptide which inhibits activity of a PKC isozyme or anactivator peptide which promotes activity of a PKC isozyme.
 3. Thecomposition of claim 1, wherein the intracellular carrier peptide isacylated at its N-terminal end.
 4. The composition of claim 1, whereinthe C-terminal arginine of the intracellular carrier peptide is aprimary carboxamide.
 5. The composition of claim 1, wherein the PKCmodulatory peptide is modified by either acylation at its N-terminalend, or amidation at its C-terminal end, or by both acylation at itsN-terminal end and amidation at its C-terminal end.
 6. The compositionof claim 1, wherein the PKC modulatory peptide is covalently linked tothe intracellular carrier peptide through the sulfhydryl group of thecysteine analog residue of the intracellular carrier peptide.
 7. Thecomposition of claim 1, which further comprises a second carrierpeptide.